Liver targeting urolithin A liposome nano-particles as well as preparation method and application thereof

Abstract

The invention discloses liver targeting urolithin A liposome nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biological medicines and nano preparations. The urolithin A liposome nanoparticle is composed of a liposome inner core, a targeting head group and an Arabic gum layer, the average particle size is in a range of 150-200nm, the liposome inner core is formed by encapsulating urolithin A by a lipid bilayer formed by soybean phosphatidylcholine and beta-stigmasterol, the targeting head group is MOTS-c and is inserted into the liposome inner core, and the targeting head group is an Arabic gum layer. The arabic gum layer is coated outside the liposome core and the targeting head group. The invention has the following beneficial effects: (1) synergistic targeting and treatment; (2) high-efficiency delivery of double drugs; (3) the stability and bioavailability are excellent; (4) the liver targeting is clear; and (5) an innovative preparation process.

Description

Technical Field

[0001] This invention relates to a nanoparticle, its preparation method and application, specifically to a liver-targeting urolithin A liposome nanoparticle, its preparation method and application, belonging to the field of biomedicine and nanoparticle technology. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. Its spectrum ranges from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH), and can further develop into liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Currently, there are no approved drugs for the treatment of NAFLD, making the development of safe and effective intervention strategies an urgent priority.

[0003] Urolithiasis A (UroA) is a major active metabolite of ellagitannins produced by gut microbiota metabolism, possessing excellent anti-inflammatory, antioxidant, mitophagy-activating, and metabolic disorder-improving biological activities. However, UroA itself is water-insoluble, chemically unstable, and has extremely low oral bioavailability (typically below 5%), which severely limits its application. MOTS-c is a 15-amino acid bioactive peptide encoded by mitochondrial DNA, with the sequence MLRQMRTSHFQVLRA. Recent studies have shown that MOTS-c can directly interact with Bcl-2 protein in hepatocytes, increasing Bcl-2 protein stability and inhibiting its ubiquitination, thereby alleviating hepatocyte apoptosis, inflammation, and steatosis, and reversing NASH progression. However, as a peptide, MOTS-c also faces the problems of easy degradation after oral administration and a short in vivo half-life.

[0004] Therefore, developing a liposomal nanoparticle that can simultaneously and efficiently deliver UroA and MOTS-c and exert their synergistic therapeutic effect on NAFLD has significant clinical implications and application value. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings of the prior art and provide urolithiasis A (UroA) liposome nanoparticles with high encapsulation efficiency, good stability, high oral bioavailability, and active liver-targeting function.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned urolithin A (UroA) liposome nanoparticles that is simple to process, has mild conditions, and is easy to scale up for production.

[0007] Another objective of this invention is to elucidate the application of the above-mentioned urolithin A (UroA) liposome nanoparticles in the prevention or treatment of non-alcoholic fatty liver disease (NAFLD), and to provide a scientific basis for its development in the pharmaceutical field.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A liver-targeting urolithin A liposome nanoparticle, comprising a liposome core, a targeting head, and a gum arabic layer, with an average particle size in the range of 150-200 nm. The liposome core is composed of a lipid bilayer encapsulating urolithin A formed by soybean phosphatidylcholine and β-stigmasterol. The targeting head is MOTS-c with the amino acid sequence MLRQMRTSHFQVLRA, inserted into the liposome core. The gum arabic layer covers the liposome core and the targeting head. The mass ratio of soybean phosphatidylcholine, β-stigmasterol, urolithin A, MOTS-c, and gum arabic is 35-40:7.5-10:4-6:4-6:50-75.

[0009] Preferably, the mass ratio of soybean phosphatidylcholine, β-stigmasterol, urolithin A, MOTS-c, and gum arabic is 36:9:5:5:60.

[0010] The aforementioned method for preparing liver-targeting urolithin A liposome nanoparticles includes the following steps: (1) Synthesis of DSPE-PEG2000-MOTS-c; (2) Prepare a stock solution of urolithiasis A with a concentration in the range of 6.0-10.0 mg / mL; (3) Accurately weigh soybean phosphatidylcholine, β-stigmasterol and DSPE-PEG2000-MOTS-c, dissolve them in an organic solvent, and form a uniform lipid film by rotary evaporation at 45°C. (4) The lipid membrane was hydrated with PBS buffer at 60°C for 45 min; (5) After hydration, add urolithin A stock solution, adjust the pH to neutral, homogenize under high pressure to obtain drug-loaded and MOTS-c modified liposomes; (6) Add gum arabic solution to the above liposomes and coat them with magnetic stirring at room temperature to obtain a suspension of urolithin A liposome nanoparticles.

[0011] Preferably, in step (1), the method for synthesizing DSPE-PEG2000-MOTS-c is as follows: (i) MOTS-c was synthesized using the standard Fmoc solid-phase synthesis method, and a cysteine ​​residue was introduced at its N-terminus to form Cys-MOTS-c; (ii) Mix DSPE-PEG2000-maleimide and Cys-MOTS-c at a molar ratio of 1:1.2 in nitrogen-protected PBS buffer and react at 4°C for 12 h. (iii) After the reaction was completed, unreacted free peptides were removed by dialysis and ultrafiltration centrifugation, and the resulting white flocculent solid DSPE-PEG2000-MOTS-c was obtained by lyophilization.

[0012] Preferably, the method for preparing urolithin A stock solution is as follows: dissolving urolithin A in a NaOH solution with a pH of 11.0-12.5.

[0013] Preferably, in step (5), the high-pressure homogenization is performed three times at 85.0 MPa.

[0014] Preferably, in step (6), the magnetic stirring time is 2 hours.

[0015] The aforementioned application of liver-targeting urolithin A liposome nanoparticles in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease.

[0016] Preferably, the drug is administered orally.

[0017] The advantages of this invention are: (1) Synergistic targeting and therapy: MOTS-c not only targets the head base and guides nanoparticles to the liver, but it is also a therapeutic drug. It works synergistically with UroA in inhibiting hepatic steatosis, inflammation and improving mitochondrial function. (2) Dual drug delivery: A single nanocarrier simultaneously encapsulates UroA and covalently bound MOTS-c, ensuring that both active ingredients, UroA and MOTS-c, can reach the site of action together, thus solving the problem of inconsistent pharmacokinetic behavior when physically mixed for drug delivery; (3) Excellent stability and bioavailability: The gum arabic (GA) coating layer effectively protects the drug-loaded liposomes, allowing them to pass smoothly through the stomach and be absorbed in the intestines, significantly improving the oral bioavailability of the two active ingredients, UroA and MOTS-c. (4) Clear liver targeting: In vitro cell uptake experiments and in vivo tissue distribution experiments showed that DSPE-PEG2000-MOTS-c modification can significantly improve the enrichment of nanoparticles in the liver and enhance the therapeutic effect. (5) Innovative preparation process: Pre-synthesized DSPE-PEG2000-MOTS-c is used to directly participate in liposome assembly. The process is simple, reproducible, and suitable for industrial production. Attached Figure Description

[0018] Figure 1 This is a scanning electron microscope image of UroA-LP-MOTS-c-GA; Figure 2 The graph shows the fluorescence intensity analysis results of DiI-LP-GA and DiI-LP-MOTS-c-GA. Figure 3 This is a graph showing the peak area changes of UroA in each group at different dosing times; Figure 4 This is a graph showing the peak area changes of UroA sulfate in each group at different administration times; Figure 5 This is a graph showing the peak area changes of UroA glucuronide in each group at different administration times. Figure 6 This is a comparison of the area under the curve (AUC) of UroA in each group 12 hours after drug administration. Figure 7 This is a comparison of the area under the curve (AUC) of UroA sulfate in each group 12 hours after drug administration. Figure 8 This is a comparison of the area under the curve (AUC) of UroA glucuronide in each group 12 hours after administration. Figure 9 This is a comparison of the total area under the curve for each group 12 hours after drug administration; Figure 10 This is a graph showing the weight changes in each group after 12 weeks of drug intervention; Figure 11 This is a comparison chart of serum triglyceride (TG) levels in each group after 12 weeks of drug intervention; Figure 12 This is a comparison chart of serum total cholesterol (TC) levels in each group after 12 weeks of drug intervention; Figure 13 This is a comparison chart of serum aspartate aminotransferase (AST) levels in each group after 12 weeks of drug intervention; Figure 14 This is a comparison chart of serum alanine aminotransferase (ALT) levels in each group after 12 weeks of drug intervention. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0020] I. Structure of Urolithin A Liposome Nanoparticles

[0021] The urolithin A liposome nanoparticles provided by this invention consist of a liposome core, a targeting head, and a gum arabic layer, with an average particle size in the range of 150-200 nm. The liposome core is composed of a lipid bilayer encapsulating urolithin A formed by soybean phosphatidylcholine and β-stigmasterol. The targeting head is MOTS-c, with the amino acid sequence MLRQMRTSHFQVLRA (SEQ ID NO: 1), inserted into the liposome core. The gum arabic layer covers the outside of the liposome core and the targeting head. The mass ratio of soybean phosphatidylcholine, β-stigmasterol, urolithin A, MOTS-c, and gum arabic is 35-40:7.5-10:4-6:4-6:50-75. Preferably, the mass ratio of soybean phosphatidylcholine, β-stigmasterol, urolithin A, MOTS-c, and gum arabic is 36:9:5:5:60.

[0022] II. Preparation of Urolithin A Liposome Nanoparticles (Comparative Example 1)

[0023] Example 1 1. Synthesis of DSPE-PEG2000-MOTS-c MOTS-c (sequence: MLRQMRTSHFQVLRA) was synthesized using the standard Fmoc solid-phase synthesis method, and a cysteine ​​residue was introduced at its N-terminus to form Cys-MOTS-c.

[0024] DSPE-PEG2000-maleimide and Cys-MOTS-c were mixed at a molar ratio of 1:1.2 in nitrogen-protected PBS buffer (pH 7.0, containing 1 mM EDTA) and reacted at 4°C for 12 h.

[0025] After the reaction was completed, unreacted free peptides were removed by dialysis and ultrafiltration centrifugation, and the resulting white flocculent solid DSPE-PEG2000-MOTS-c was obtained by lyophilization.

[0026] Identification by MALDI-TOF mass spectrometry: Molecular weight calculated [M+H] + The measured value was 4582.3, while the actual value was 4582.8, indicating a successful connection.

[0027] 2. Preparation of UroA stock solution Dissolve UroA in a NaOH solution with a pH of 12.0 (or within the range of 11.0-12.5) to prepare a stock solution of UroA with a concentration of 8.0 mg / mL (or within the range of 6.0-10.0 mg / mL).

[0028] 3. Preparation of drug-loaded liposomes Accurately weigh 72 mg of soybean phosphatidylcholine, 18 mg of β-stigmasterol and 10 mg of DSPE-PEG2000-MOTS-c, dissolve them in 10 mL of organic solvent chloroform, and form a uniform lipid film by rotary evaporation at 45 °C.

[0029] The lipid film was hydrated with 5 mL of PBS buffer (pH 7.4) at 60 °C for 45 min.

[0030] After hydration, 1.25 mL of UroA stock solution was added, and the pH was adjusted to neutral (7.0) with 0.1 M HCl. The mixture was homogenized three times under high pressure at 85.0 MPa to obtain drug-loaded (UroA) and MOTS-c modified liposomes, denoted as UroA-LP-MOTS-c.

[0031] 4. Coat with gum arabic Add 6 mL of 20 mg / mL gum arabic (GA) solution to the above liposome UroA-LP-MOTS-c and gently stir magnetically for 2 h at room temperature for coating. After coating, a suspension of urolithin A liposome nanoparticles is obtained, denoted as UroA-LP-MOTS-c-GA, and stored in a refrigerator at 4°C for later use.

[0032] The characterization results of UroA-LP-MOTS-c-GA are as follows: (1) Average particle size: 168±4nm (scanning electron microscope results are shown in the figure) Figure 1 ); (2) Polydispersity Index (PDI): 0.19; (3) Zeta potential: -30.5±1.2mV; (4) UroA encapsulation efficiency: 93.2±1.6%.

[0033] Comparative Example 1

[0034] The preparation method is basically the same as in Example 1, except that DSPE-PEG2000-MOTS-c is not added.

[0035] The prepared urolithin A liposome nanoparticle suspension was designated UroA-LP-GA and stored at 4°C for later use. Comparative Example 2

[0036] The preparation method is basically the same as in Example 1, except that DSPE-PEG2000-MOTS-c is replaced with DSPE-PEG2000-SP (Scramble Peptide) of equal mass.

[0037] Preparation method of DSPE-PEG2000-SP: SP (sequence: ARQVLHSFMRLTRQM, SEQ ID NO: 2) was synthesized using the standard Fmoc solid-phase synthesis method, and a cysteine ​​residue was introduced at its N-terminus to form Cys-SP. DSPE-PEG2000-maleimide (1 equiv.) and Cys-SP (1.2 equiv.) were mixed in nitrogen-protected PBS buffer (pH 7.0, containing 1 mM EDTA) and reacted at 4 °C for 12 h. After the reaction, unreacted free peptides were removed by dialysis and ultrafiltration centrifugation. The resulting white flocculent solid, DSPE-PEG2000-SP, was obtained by lyophilization. MALDI-TOF mass spectrometry identification: Molecular weight [M+H] was calculated. + The measured value was 4582.3, which proves that the connection was successful.

[0038] The prepared urolithin A liposome nanoparticle suspension was designated as UroA-LP-SP-GA and stored at 4°C for later use. Comparative Example 3

[0039] UroA and MOTS-c were physically mixed at a mass ratio of 1:1.

[0040] The resulting product is designated as UroA-MOTS-c.

[0041] III. Evaluation of the performance of UroA-LP-MOTS-c-GA 1. Evaluation of in vitro cell uptake and targeting Urolithin A liposome nanoparticles UroA-LP-GA and UroA-LP-MOTS-c-GA were labeled with DiI (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindocyanine perchlorate) as a fluorescent probe to investigate their in vitro targeting properties.

[0042] Methods for DiI labeling of UroA-LP-MOTS-c-GA: Accurately weigh 72 mg of soybean phosphatidylcholine, 18 mg of β-stigmasterol, 10 mg of DSPE-PEG2000-MOTS-c, and 0.36 mg of DiI, dissolve them in 10 mL of chloroform, and evaporate them by rotary evaporation at 45 °C to form a uniform lipid film. Then, hydrate the lipid film with 5 mL of PBS buffer (pH 7.4) at 60 °C for 45 min. After hydration, add 1.25 mL of UroA stock solution, adjust the pH to neutral (7.0) with 0.1 M HCl, and homogenize three times under high pressure at 85.0 MPa. Then, coat the lipid film with gum arabic in vitro using the same method as in Example 1. Finally, remove the free DiI by dialysis (molecular weight cutoff 8000-14000 Da). The DiI-labeled UroA-LP-MOTS-c-GA is denoted as DiI-LP-MOTS-c-GA.

[0043] Methods for DiI labeling of UroA-LP-GA: The method is basically the same as that of DiI-labeled UroA-LP-MOTS-c-GA, the only difference being that DSPE-PEG2000-MOTS-c is not added. UroA-LP-GA labeled with DiI is recorded as DiI-LP-GA.

[0044] Normally cultured HepG2 cells were digested with 5% (w / v) trypsin and then the cell density was adjusted to 1×10⁻⁶ cells using complete culture medium. 5 Cells were cultured at a density of 1 / mL and then transferred to 6-well plates at 1 mL per well. The plates were incubated in a 5% CO2 incubator for 24 h. After incubation, the culture medium was discarded, and 1.0 mL of DiI-LP-GA (diluted 20-fold with serum-free fetal bovine serum) or DiI-LP-MOTS-c-GA (diluted 20-fold with serum-free fetal bovine serum) was added. The plates were then incubated in a 5% CO2 incubator for another 4 h. After incubation, the supernatant was discarded, and the cells were washed once with pre-chilled PBS buffer (pH 7.0) at 4°C. The cells were then digested with 5% (w / v) trypsin, centrifuged at 1000 rpm, and the supernatant was discarded. The cells were resuspended in PBS buffer (pH 7.0). After centrifugation again, the supernatant was discarded, and the cells were resuspended in PBS buffer (pH 7.0) to form a single-cell suspension. Fluorescence intensity was analyzed using flow cytometry. The experiment was repeated three times.

[0045] The fluorescence intensity analysis results of DiI-LP-GA and DiI-LP-MOTS-c-GA are shown in the figure. Figure 2 .

[0046] exist Figure 2 Based on this, the mean fluorescence intensity (MFI) of DiI-LP-GA and DiI-LP-MOTS-c-GA was calculated using ImageJ. The results are as follows: The MFI of DiI-LP-MOTS-c-GA was 5360±240, while that of DiI-LP-GA was 1750±190. The cellular uptake of DiI-LP-MOTS-c-GA was 3.06 times that of DiI-LP-GA (p<0.01).

[0047] It is evident that MOTS-c modification significantly enhances the uptake of liposomes in liver cells.

[0048] 2. Study in vivo pharmacokinetics

[0049] Forty male SD rats (weighing 220g±20g) were randomly divided into four groups: free UroA group (administered commercially available pure UroA), UroA-LP-GA group (administered suspension UroA-LP-GA prepared in Comparative Example 1), UroA-MOTS-c group (administered product UroA-MOTS-c prepared in Comparative Example 3), and UroA-LP-MOTS-c-GA group (administered suspension UroA-LP-MOTS-c-GA prepared in Example 1), with 10 rats in each group. All groups were administered UroA at a dose of 20mg / kg by gavage.

[0050] Blood samples (0.5 mL) were collected from rats via tail vein at 1.0 h, 2.0 h, 3.0 h, 4.0 h, 6.0 h, 8.0 h, 10.0 h, and 12.0 h after drug administration. The samples were centrifuged at 4000 rpm for 15 min, and the supernatant was collected to obtain serum samples. Then, 300 μL of methanol was mixed with 100 μL of serum sample, vortexed for 5 min, and then centrifuged at 15000 rpm for 10 min. The supernatant was collected, dried under nitrogen, and reconstituted with 100 μL of methanol. The concentration of UroA in the samples was determined by HPLC-MS / MS. After the experiment, all rats were euthanized by carbon dioxide inhalation.

[0051] The pharmacokinetic results of UroA-LP-MOTS-c-GA in rats are shown in the table below. Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 .

[0052] Depend on Figures 3 to 9It can be seen that: compared with the free UroA group, there was no significant difference in the oral bioavailability of UroA in the UroA-MOTS-c group, but the UroA-LP-GA group and the UroA-LP-MOTS-c-GA group could significantly improve the oral bioavailability of UroA, and the UroA-LP-MOTS-c-GA group was significantly higher than that of the UroA-LP-GA group.

[0053] 3. Evaluate the effect of UroA-LP-MOTS-c-GA in alleviating NAFLD.

[0054] Sixty male C57BL / 6 mice (8 weeks old, 22±2g) were housed under standard light conditions at 21-24℃. After one week of acclimatization, the mice were randomly divided into six groups: normal group, model group, UroA-MOTS-c group (UroA-MOTS-c prepared by comparison example 3), UroA-LP-GA group (UroA-LP-GA suspension prepared by comparison example 1), UroA-LP-SP-GA group (UroA-LP-SP-GA suspension prepared by comparison example 2), and UroA-LP-MOTS-c-GA group (UroA-LP-MOTS-c-GA suspension prepared by comparison example 1), with 10 mice in each group.

[0055] The normal group was fed a regular diet, while the other 5 groups were fed a high-fat diet (14% protein, 26% carbohydrates, 60% fat). The NAFLD model was established in 8 weeks.

[0056] Except for the normal group and the model group, all groups were administered UroA at a dose of 20 mg / kg by gavage, while the normal group and the model group were administered an equal volume of distilled water by gavage.

[0057] During the rearing period, the mice had free access to water. The drug intervention lasted 12 weeks, and weight changes were recorded weekly. See below for details of weight changes. Figure 10 .

[0058] Depend on Figure 10It was found that the weight of mice in the normal group increased steadily throughout the intervention period. Compared with the normal group, the weight of mice in the model group (high-fat diet) showed a significant and rapid increase from week 2, indicating that the NAFLD model was successfully established. Weight gain in all treatment groups was inhibited to varying degrees. The UroA-MOTS-c group showed only a slight reduction in weight gain, while the UroA-LP-GA and UroA-LP-SP-GA groups showed more significant inhibitory effects with no statistically significant difference between the two groups. The weight gain curve of the UroA-LP-MOTS-c-GA group was closest to that of the normal group, with a significantly lower weight gain than the model group and other treatment groups, indicating that UroA-LP-MOTS-c-GA has the best efficacy in improving metabolism and controlling weight gain.

[0059] All mice were fasted for at least 12.0 h before blood samples were collected. After euthanasia of the mice by carbon dioxide inhalation, blood samples were obtained through the ophthalmic venous plexus. The samples were centrifuged at 4000 rpm for 15 min at 4 °C, and the supernatant was collected to obtain serum samples, which were stored at -80 °C for later testing.

[0060] The results of liver function analysis of each group of mice are shown below. Figure 11 , Figure 12 , Figure 13 and Figure 14 .

[0061] Depend on Figures 11 to 14 It can be seen that, compared with the model group, each group showed a certain efficacy in alleviating NAFLD. Among them, the UroA-LP-MOTS-c-GA group had the best efficacy, followed by the UroA-LP-GA group and the UroA-LP-SP-GA group, with no significant difference between the two groups. The UroA-MOTS-c group had the weakest efficacy.

[0062] It is evident that the MOTS-c specific sequence also has a therapeutic effect on NAFLD.

[0063] In summary, this invention successfully prepared a high-performance MOTS-c-modified liver-targeted UroA delivery system. Through the synergistic effect of DSPE-PEG2000-MOTS-c and gum arabic, efficient co-delivery of UroA and MOTS-c was achieved, demonstrating excellent liver targeting and synergistic therapeutic effects, laying a solid foundation for the development of NAFLD intervention products based on UroA and MOTS-c.

[0064] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A liver-targeted urolithin A liposome nanoparticle, characterized in that, The urapidil liposome nanoparticle is composed of a liposome core, a targeting head group and an acacia layer, and has an average particle size in the range of 150-200 nm, wherein the liposome core is composed of a lipid bilayer of soybean phosphatidylcholine and beta-stigmasterol encapsulating urapidil, the targeting head group is MOTS-c with an amino acid sequence of MLRQMRTSHFQVLRA, which is inserted into the liposome core, and the acacia layer is coated on the outside of the liposome core and the targeting head group, and the mass ratio of soybean phosphatidylcholine, beta-stigmasterol, urapidil, MOTS-c and acacia is 35-40:7.5-10:4-6:4-6:50-75.

2. The liver-targeting urolithin A liposome nanoparticle according to claim 1, wherein, The mass ratio of soybean phosphatidylcholine, beta-stigmasterol, urapidil, MOTS-c and acacia is 36:9:5:5:

60.

3. The method of claim 1, wherein the liver-targeting urolithin A liposome nanoparticle is prepared by, The method comprises the following steps: (1) synthesizing DSPE-PEG2000-MOTS-c; (2) preparing a urapidil stock solution with a concentration in the range of 6.0-10.0 mg / mL; (3) accurately weighing soybean phosphatidylcholine, beta-stigmasterol and DSPE-PEG2000-MOTS-c, dissolving them in an organic solvent, and forming a uniform lipid film at 45°C through rotary evaporation; (4) hydrating the lipid film with PBS buffer at 60°C for 45 min; (5) after hydration, adding the urapidil stock solution, adjusting the pH to neutral, and obtaining the drug-loaded MOTS-c modified liposome through high-pressure homogenization; (6) adding an acacia solution to the above liposome, and coating it through gentle magnetic stirring at room temperature to obtain a urapidil liposome nanoparticle suspension.

4. The preparation method of liver-targeting urolithin A liposome nanoparticles according to claim 3, characterized in that, In step (1), the method for synthesizing DSPE-PEG2000-MOTS-c is as follows: (i) synthesizing MOTS-c by using a standard Fmoc solid-phase synthesis method, and introducing a cysteine residue at the N-terminus to form Cys-MOTS-c; (ii) mixing DSPE-PEG2000-maleimide and Cys-MOTS-c in a molar ratio of 1:1.2 in a PBS buffer under nitrogen protection, and reacting at 4°C for 12 h; (iii) after the reaction is completed, removing the unreacted free peptides through dialysis and ultrafiltration centrifugation, and obtaining white flocculent solid DSPE-PEG2000-MOTS-c after freeze-drying.

5. The preparation method of liver-targeting urolithin A liposome nanoparticles according to claim 3, characterized in that, In step (2), the method for preparing the urapidil stock solution is as follows: dissolving urapidil in a NaOH solution with a pH of 11.0-12.

5.

6. The preparation method of liver-targeting urolithin A liposome nanoparticles according to claim 3, characterized in that, In step (5), high-pressure homogenization is performed 3 times at 85.0 MPa.

7. The method for preparing liver-targeted urolithin A liposome nanoparticles according to claim 3, characterized in that, In step (6), the magnetic stirring time is 2 h.

8. The use of the liver-targeting urapidil liposome nanoparticle of claim 1 in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

9. Use according to claim 8, characterized in that, The drug is administered through the oral route.

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